Bone sialoprotein (BSP) is a noncollagenous component of bone extracellular matrix (ECM) considered to play a fundamental role in biomineralization. This proposal will establish the nuclear mechanism regulating BSP expression in bone and define its role in mineralization by achieving the following specific aims: Aim 1. Define 5'-regulatory regions of the BSP gene and related nuclear proteins necessary for bone and ECM-specific expression. The applicant proposes that signals provided by the ECM are essential for tissue-specific expression of BSP and that ECM regulates BSP and other osteoblast-related gene products through a common nuclear mechanism involving the activation/induction of specific nuclear proteins. The mouse BSP gene promoter and/or intronic sequences will be used to define DNA sequences and associated nuclear proteins necessary for ECM-dependent and tissue-specific expression. Aim 2. Establish the role of BSP in osteoblast-mediated biomineralization. BSP has several properties consistent with it playing an important role in the initiation of mineralization including ability to nucleate hydroxyapatite crystal formation in vitro. However, its role in mineralization has not yet been examined in a bone-like environment. Using an osteoblast cell culture system that forms and mineralizes a bone-like ECM, planned studies will use multiple approaches to elucidate the role of BSP in mineralization including antibody blocking experiments, antisense RNAs and forced expression. Site directed mutagenesis will then define critical regions of the BSP molecule necessary for activity. This project will identify a common mechanism whereby osteoblast ECM controls the tissue-specific expression of BSP and other osteoblast proteins essential knowledge for understanding pathological conditions that disrupt matrix integrity or synthesis such as periodontal disease, rheumatoid arthritis, osteogenesis imperfecta and osteoporosis. By defining the role of BSP in physiological mineralization studies will resolve a long standing controversy concerning the role/s of this phosphoprotein in bone formation develop an approach that could be applied to understanding the function of other related proteins in bones and teeth, and provide a means for regulating mineralization in diseased states.